Structure /Function of Phospholamban in Heart

磷脂班在心脏中的结构/功能

• 批准号: 6677773
• 负责人: LARRY R. JONES
• 金额: $ 44万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6677773
• 关键词: active sites; biological signal transduction; calcium transporting ATPase; chemical models; crosslink; crystallization; dogs; enzyme complex; enzyme inhibitors; genetic manipulation; heart contraction; intermolecular interaction; molecular biology information system; monomer; nucleotides; phospholamban; phosphorylation; protein binding; protein protein interaction; protein sequence; protein structure function; sarcoplasmic reticulum; thapsigargin; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): The long term goal of this research is to elucidate the molecular and biophysical mechanism by which phospholamban (PLB) inhibits the activity of the Ca pump (SERCA2a isoform) in cardiac sarcoplasmic reticulum (SR). PLB is a pentameric phosphoprotein in cardiac SR, which is composed of five identical monomers. Previously, we demonstrated the PLB monomer is responsible for binding to SERCA2a and inhibiting it. Now, we propose to localize the binding-interaction sites between the PLB monomer and SERCA2a that lead to enzyme inhibition, and determine how the molecular interaction is regulated by key allosteric modulators including Ca concentration, nucleotides, and phosphorylation. Emphasis will be placed upon identifying amino acids in the inhibitory complex that interact directly, taking advantage of our newly developed chemical cross-linking method. In Aim 1, we will perform Cys-scanning mutagenesis of PLB to localize distinct sites along its primary structure that cross-link to endogenous Cys residues of SERCA2a. The cross-linked Cys residues of SERCA2a will be directly identified by protein purification/peptide sequencing. In Aim 2, Lys residues of SERCA2a that cross-link to distinct sites of PLB will be localized. By use of crosslinking agents as molecular rulers and combining results from Aims 1 and 2, we will develop an accurate 3-D model of the binding-complex formed between the PLB monomer and SERCA2a. In Aim 3, the effects of Ca concentration, nucleotides, and the inhibitor thapsigargin on cross-linking of PLB to SERCA2a will be investigated. The hypothesis tested is that PLB binds exclusively to the Ca-free form (E2) of SERCA2a, but only that E2 state that has bound ATP or ADP. In Aim 4, we will determine how Ca relieves PLB inhibition of SERCA2a. We hypothesize that PLB binds preferentially to E2, antagonizing Ca binding to SERCA2a, and that Ca binds preferentially to El, antagonizing PLB binding to SERCA2a. The Ca-binding site of SERCA2a responsible for dissociating PLB from the pump will be identified, and the effect of PLB on the Ca-binding affinity of SERCA2a will be quantified. In Aim 5, we will determine how phosphorylation of PLB by protein kinases relieves PLB inhibition. The hypothesis tested is that phosphorylation of PLB directly dissociates it from SERCA2a. Here we will also determine if the two phosphorylated residues of PLB, Ser 16 and Thr 17, interact directly with SERCA2a to aid in enzyme inhibition. PLB is a key regulator of myocardial contractile dynamics. By defining its molecular mechanism of action on the Ca pump, new insights on PLB regulation of the strength of the heartbeat will result, that may ultimately lead to the design of new drugs to treat heart failure.

描述(申请人提供)：这项研究的长期目标是阐明磷蛋白(PLB)抑制心肌肌浆网(SR)钙泵(SERCA2a亚型)活性的分子和生物物理机制。PLB是心脏SR中的一种五聚体磷酸蛋白，由5个相同的单体组成。之前，我们证明了PLB单体负责与SERCA2a结合并抑制它。现在，我们建议定位PLB单体与SERCA2a之间导致酶抑制的结合作用部位，并确定关键的变构调节剂(包括钙浓度、核苷酸和磷酸化)如何调节分子相互作用。重点将放在识别抑制复合体中直接相互作用的氨基酸上，利用我们新开发的化学交联法。在目标1中，我们将对PLB进行半胱氨酸扫描突变，沿着其一级结构定位与SERCA2a内源性半胱氨酸残基交联的不同位点。SERCA2a的交联型半胱氨酸残基将通过蛋白质纯化/多肽测序直接鉴定。在目标2中，将定位SERCA2a的Lys残基，该残基与PLB的不同位点发生交联。通过使用交联剂作为分子尺子，结合AIMS 1和2的结果，我们将建立PLB单体与SERCA2a之间形成的结合络合物的精确三维模型。在目标3中，将研究钙浓度、核苷酸和阻断剂thapsigargin对PLB与SERCA2a交联的影响。被检验的假设是PLB仅与SERCA2a的无钙形式(E2)结合，但只与结合了ATP或ADP的E2状态结合。在目标4中，我们将确定钙如何解除SERCA2a对原核细胞的抑制。我们假设PLB优先与E2结合，拮抗Ca与SERCA2a的结合，而Ca优先与EL结合，拮抗PLB与SERCA2a的结合。将确定负责将PLB从泵中解离的SERCA2a的钙结合部位，并将量化PLB对SERCA2a与钙结合的亲和力的影响。在目标5中，我们将确定蛋白激酶对PLB的磷酸化如何解除对PLB的抑制。被检验的假设是PLB的磷酸化直接使其与SERCA2a解离。在这里，我们还将确定PLB的两个磷酸化残基，Ser16和Thr17，是否直接与SERCA2a相互作用，以帮助酶抑制。PLB是心肌收缩动力学的关键调节因子。通过确定其在钙泵上的分子作用机制，将对PLB调节心跳强度产生新的见解，最终可能导致设计治疗心力衰竭的新药。